A number of human diseases are today treated by therapeutic monoclonal antibodies, for example humanized or fully human monoclonal antibodies. In 1975, Kohler and Milstein produced murine hybridomas that secreted monoclonal antibodies (mAbs) of defined specificity and ushered in the modern era of therapeutic mAbs utilizing these new hybridoma technologies (Kohler and Milstein, Nature 256:495-97, 1975). However, major limitations of these early therapeutics included the lack of effector function, reduced serum half-life and the increased propensity to elicit an undesired immune response. Chimerization and humanization technologies helped to overcome these unwanted characteristics (Carson and Friemark, Adv. Immunol. 38:275-311, 1986; James et al., Scottish Med. J. 29: 67-83, 1984; Morrison, Science 229:1202-1207, 1985).
First generation bispecific antibodies (BsmAbs) that were produced by fusing two established hybridoma cell lines together to form a hybrid hybridoma or quadroma (Milstein and Cuello, Nature 305:537-540, 1983) or by chemical crosslinking two F(ab′) fragments (Karpovsky et al., J. Exp. Medicine 160: 1686-1701, 1984) allowed simultaneous modulation of multiple targets. Although these studies highlighted the therapeutic potential of BsmAbs, these approaches, in addition to adverse in vivo responses to murine antibody fragments, presented logistical problems with respect to producing large, homogenous lots of purified antibodies. For example, random association of heavy and light chains secreted by the hybrid hybridomas results in production of 10 different antibody species from which the desired bispecific molecule should be isolated.
The first humanized BsmAb, MDX-447, (Curnow, Cancer Immunol. Immunother. 45:210-215, 1997) was generated by CDR-grafting followed by chemical coupling of the two Fab′ domains to create a bispecific F(ab′)2 molecule. However, the process of reduction, oxidation and subsequent purification underscores the key hurdle in generating highly pure BsmAb molecules from the employment of these methods. Isolation and purification of the heterodimeric species from a homodimeric species is not possible at production scale (Karacay et al., Bioconjugate Chem. 11:842-854, 2000).
Additional BsmAb platforms have been developed including diabodies (Holliger et al., Proc. Natl. Acad. Sci. 90: 6444-6448, 1993), single-chain diabodies (Brusselbach et al., Tumor Targeting 4:115-123, 1999; Nettlebeck et al., Molecular Therapy 3:882-891, 2001), tandem single-chain variable fragments (scFv) (Bi-specific T-cell engagers (BiTEs)) (Mack et al., Proc. Natl. Acad. Sci. 92:7021-7025, 1995.), knob and hole mAbs (Ridgeway et al., Protein Engineering 9:617-621, 1996.), and dual variable domain antibodies (WO2007/024715).
There is a need in the art for improved binding proteins capable of binding at least one target and providing ease of manufacturing and reduced cost of goods.